This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding tyrosine biosynthetic enzymes in plants and seeds.
Animals do not synthesize aromatic amino acids so it is necessary to include them in their diets. In the aromatic amino acid biosynthetic pathway chorismate is converted to anthranilate during tryptophan biosynthesis and it is converted to prephenate, the branch point for tyrosine and phenylalanine biosynthesis. Chorismate mutase catalyzes the conversion of chorismate to prephenate. Two different isoforms of chorismate mutase have been identified. A chorismate mutase located in the chloroplasts (CM-1) is activated by tryptophan and inhibited by phenylalanine and tyrosine while a cytoplasmic chorismate mutase (CM-2) is insensitive to the presence of all three aromatic amino acids (Singh et al. (1985) Arch. Biochem. Biophys. 243:374-384).
Prephenic acid is converted to tyrosine either by a) oxidative decarboxylation catalyzed by prephenate dehydrogenase followed by transamination catalyzed by aromatic aminotransferase or by b) transamination of prephenate catalyzed by prephenate aminotransferase followed by oxidative decarboxylation catalyzed by arogenate dehydrogenase. Arogenate dehydrogenase activity is commonly found in plants while prephenate dehydrogenase activity has been difficult to detect.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 62 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn chorismate mutase polypeptide of SEQ ID NOs:2 and 12, a rice chorismate mutase polypeptide of SEQ ID NOs:4 and 14, a soybean chorismate mutase polypeptide of SEQ ID NOs:6 and 16, a wheat chorismate mutase polypeptide of SEQ ID NOs:8 and 18. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 60 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a soybean prephenate dehydrogenase polypeptide of SEQ ID NOs:10 and 20. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, and 20. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a chorismate mutase polypeptide of at least 62 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 12, 14, 16,and 18.
The present invention relates to a prephenate dehydrogenase polypeptide of at least 60 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10 and 20.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a chorismate mutase or a prephenate dehydrogenase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level a chorismate mutase or a prephenate dehydrogenase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of the chorismate mutase or the prephenate dehydrogenase polypeptide in the host cell containing the isolated polynucleotide with the level of the chorismate mutase or the prephenate dehydrogenase polypeptide in the host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a chorismate mutase or a prephenate dehydrogenase polypeptide gene, preferably a plant chorismate mutase or prephenate dehydrogenase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a chorismate mutase or a prephenate dehydrogenase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a chorismate mutase or a prephenate dehydrogenase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a horismate mutase or a prephenate dehydrogenase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a horismate mutase or a prephenate dehydrogenase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of chorismate mutase or prephenate dehydrogenase in the transformed host cell; (c) optionally purifying the chorismate mutase or the prephenate dehydrogenase expressed by the transformed host cell; (d) treating the chorismate mutase or the prephenate dehydrogenase with a compound to be tested; and (e) comparing the activity of the chorismate mutase or the prephenate dehydrogenase that has been treated with a test compound to the activity of an untreated chorismate mutase or prephenate dehydrogenase, thereby selecting compounds with potential for inhibitory activity.
The present invention relates to a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.
The present invention relates to an isolated polynucleotide of the present invention comprising at least 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.
The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.
The present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably plant cell, such as a monocot or a dicot, under conditions which allow expression of the tyrosine biosynthetic enzyme polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.